Immobilization of enzymes with a cross-linking agent and a polymer containing l-amino ethylene moieties

ABSTRACT

Physically strong particles containing an immobilized enzyme are prepared for use in a fixed bed-column. The particles are prepared by adding a homopolymer of 1-amino ethylene or a copolymer of 1-amino ethylene and N-vinyl formamide to an aqueous medium containing an enzyme, adding a cross-linking agent to cross-link the enzyme and polymer and cause flocculation, and dewatering, sub-dividing and drying the resultant flocculant. Preferably, the enzyme is glucose isomerase and the cross-linking agent is glutaraldehyde, polyazetidine or diisocyanate.

TECHNICAL FIELD

This invention relates to a particulate immobilized enzyme preparation,to use of the immobilized enzyme preparation in an enzyme-catalyzedprocess and to a process for immobilizing enzymatically activebiological material.

BACKGROUND ART

It is known that enzymes can be immobilized without using a carrier bycross-linking with polyethylene imine (PEI) and a cross-linking agentsuch as glutaraldehyde or polyazetidine (e.g. U.S. Pat. No. 4,288,552,U.S. Pat. No. 4,355,105, EP 297,912). Such methods can be used toproduce immobilized enzymes in particle form with good activity andphysical strength, suitable for continuous use in a fixed-bed column.However, PEI is an expensive material, so it is desirable to findalternatives to this. Also, immobilization of some enzymes with PEI sometimes leads to poor flocculation, resulting in difficult dewatering.

It is the purpose of the invention to provide a method with improvedflocculation and dewatering and without the need for PEI for producingan immobilized enzyme preparation with satisfactory properties forfixed-bed column use: activity, stability (half life) and physicalstrength (pressure drop).

SUMMARY OF THE INVENTION

It has surprisingly been found that the object can be obtained byreplacing polyethyleneimine with a certain polymer that has never beendescribed for use in immobilization.

Accordingly, the invention provides a particulate immobilized enzymepreparation obtainable by a process comprising the sequential steps of:

a) providing an aqueous medium containing enzymatically activebiological material

b) adding a polymer containing 1-amino ethylene moieties and,optionally, N-vinyl formamide,

c) adding a cross-linking agent for amino groups,

d) holding the mixture to effect cross-linking and flocculation,

e) dewatering,

f) sub-dividing, and

g) drying.

The invention also provides use of the immobilized enzyme preparation inan enzyme-catalyzed process. Finally, the invention provides a processfor immobilizing enzymatically active biological material, characterizedby comprising the above sequential steps.

DETAILED DESCRIPTION OF THE INVENTION

The biological material to be immobilized according to the invention isenzymatically active. It may comprise enzymatically active microbialcells in the form of a culture broth containing intact cells or cellpaste consisting of partly or fully disrupted cells. The biologicalmaterial may also consist of or comprise a cell-free enzyme solution orpurified enzyme.

The biological material may also comprise inactive protein, preferably0-50% by weight. Thus, if highly purified enzyme is to be immobilized,it may be preferable to add inert protein such as albumin or gelatin.

The quantity of water present in the reaction mixture is not critical.Excess water will be removed during dewatering without any serious lossof active material. Thus, water may be added to obtain a convenientconsistency. Conveniently, the biological material is added in the formof an aqueous dispersion or solution typically with 1-25% (w/w) of drysubstance, particularly 1-10% in the case of culture broth or 10-25% inthe case of a purified enzyme.

The polymer to be used in the invention may be a homopolymer of1-aminoethylene or a copolymer of this monomer and N-vinyl formamide. Itpreferably contains 10-100 mole % (most preferably 25-50%) of --CH₂--CH(NH₂)-- units and 0-90% mole % (most preferably 50-75%) of --CH₂--CH(NH--CHO)-- units. It preferably has a molecular weight in the range50,000-500,000. Such polymers may be produced by hydrolysis ofN-vinylformamide homopolymer, e.g. according to U.S. Pat. No. 4,421,602or EP 71,050. The molecular weight of the polymer is described in thisapplication by the Fikentscher K value of the non-hydrolysedN-vinylformamide homopolymer as described in U.S. Pat. No. 4,421,602.This K value can vary between 10 and 200. The amount of polymer ispreferably 2-30% by weight of the dry matter in the biological materialand most preferably 2-15%.

Chitosan may be used in addition to the above-mentioned polymer.Preferably, 1-15% of chitosen and 1-15% of the polymer are used (% byweight of the dry matter in the biological material). The chitosanshould be introduced before the addition of cross-linking in step c); itmay be added together with the polymer.

The cross-linking agent used in the invention is one that reacts withamino groups. Examples are glutaraldehyde, diisocyanates (e.g. toluyleneor hexamethylene diisocyanate) and polyazetidine (as described in EP297,912).

Optionally, cross-linking agent may also be added to the aqueous mediumof step a) and the mixture held sufficiently long to let partialcross-linking occur (e.g. 5-20 minutes) before introducing the polymer.In this case, the ratio of cross-linking agent added in step a) to thatadded in step c) is preferably 1:2-1:4.

The total amount of cross-linking agent is preferably in the range 5-40%by weight of the dry matter in the biological material. A relativelyhigh amount, e.g. 20-40%, may be preferred in order to obtain physicallystrong particles; alternatively, a lower amount (e.g. 10-20%) may beused in order to obtain particles with less diffusion restriction andhigher activity. The holding time in step d) is preferably 0.5-2 hours.

The temperature throughout the process is generally in the range 0°-60°C. Temperature near ambient is often convenient, but lower temperaturemay be needed due to enzyme instability.

pH throughout the process is generally around neutral, mostly betweenabout 5 and about 9. Higher or lower pH may be preferred depending onthe enzyme stability. A buffer may be included to stabilize pH duringthe reaction.

The dewatering step of the invention is intended to remove excess waterafter flocculation thereby generating a pasty mass. It is convenientlydone by filtration or centrifugation.

Sub-dividing according to the invention is done to form the dewateredmass into individual particles of controlled size. A preferred techniqueis extrusion. Optionally, the extruded particles may be rounded(spheronized) before or after curing, e.g. by the "Marumerizer"technique disclosed in British patent specification GB 1,362,265.

The subdivided material is dried, e.g. to a water content below of10-25% w/w. With an ultimate water content above 25% the microbialstability of the product may be unsatisfactory, and the particles maytend to aggregate over time in storage. Drying to a water content ofbelow about 10% may inactivate the biological material. Preferredtechniques are air drying or fluidized-bed drying, generally at 15°-80°C. In case of very sensitive biological materials, low temperaturedrying or freeze-drying may be needed.

The invention may be applied to immobilization of a wide range ofenzymes. Some examples follow:

Glucose isomerase, e.g. derived from Streptomyces (especially S.murinus), Bacillus (especially B. coagulans) or Actinoplanes (especiallyA. missouriensis).

Aminopeptidase, e.g. derived from Pseudomonas

Penicillin acylase, e.g. derived from Fusarium.

Nitrilase, e.g. from Rhodococcus (especially Rh. erythropolis), fromPseudomonas or from Brevibacterium.

Fructosyl transferase, e.g. from Aspergillus.

Invertase, e.g. from Saccharomyces.

Lactase, e.g. from Kluyveromyces.

Cyanidase, e.g. from Alcaligenes.

EXAMPLES EXAMPLE 1

A reference sample was prepared as follows:

1 l of glucose isomerase containing fermentation liquid fromStreptomyces murinus (prepared according to U.S. Pat. No. 4,687,742, drysubstance content 4%), was mixed with 10 g MgSO₄ •7H₂ O and pH adjustedto 7.5. 5 ml 50% glutaraldehyde was added and the cell sludge wasstirred for 10 minutes under pH-adjustment to pH 7.5. Then 5.0% (on drymatter) of polyethylene imine (Sedipur, product of BASF, West Germany)was added and after thorough mixing 10 ml glutaraldehyde (totalglutaraldehyde 18% based on cell sludge plus polyethylene imine drysubstance) for cross-linking of the mixture. pH was constantly adjustedto 7.5. After 1 hour the cross-linked mixture was flocculated byaddition of a cationic flocculant, Superfloc C 521 (Cyanamid Int.). Thecross-linked enzyme was recovered by filtration, formed into particlesby extrusion through a 0.8 mm screen and dried at room temperature.

A series of preparations according to the invention were made asdescribed above, but with polyethyleneimine replaced by a polymer of1-amino ethylene with or without N-vinyl formamide (experimentalpreparations from BASF, Germany). The composition of the polymers isshown in Table 1.

The glucose isomerase activity was measured by Novo Analysis MethodF-855310 (available on request from Novo Nordisk A/S, Denmark). Bothinitial activity of the immobilized enzyme and in some cases theactivity during several months of continuous isomerization in laboratoryscale columns (60° C., pH 7.5) was measured. The activity decay isexpressed by the half life, the time where the activity is equal to halfof the initial activity.

The pressure drop was measured over a column with a diameter of 24 mmand an enzyme bed height of 4 cm (5 g enzyme). The solution, 45% glucosein demineralized water with 1 g MgSO₄ /l, was pumped through the columnat a rate of 40 g/min at 60° C. The pressure drop (in mm of liquid)describes the physical stability of the enzyme particle, i.e. a lowpressure drop corresponds to a good physical stability.

The experimental results (initial activity, half life and pressure drop)are shown in Table 2.

                  TABLE 1                                                         ______________________________________                                                               Molecular weight                                               Mole %         Mn                                                     Polymer --CH.sub.2 --CH(NH.sub.2)--                                                                  g/mol        K value                                   ______________________________________                                        A       67             4-5 × 10.sup.5                                                                       106                                       B       100            4-5 × 10.sup.5                                                                       106                                       C       32             2-3 × 10.sup.5                                                                       86                                        D       61             2-3 × 10.sup.5                                                                       86                                        E       95             2-3 × 10.sup.5                                                                       86                                        F       43             3-4 × 10.sup.4                                                                       32                                        G       61             3-4 × 10.sup.4                                                                       32                                        H       98             3-4 × 10.sup.4                                                                       32                                        ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                                 Glucose isomerase                                                                           Half life Pressure drop                                Polymer  activity IGIU/g                                                                             hours     g/cm.sup.2                                   ______________________________________                                        Reference                                                                              350           2000      15                                           A        450           1000      50                                           B        300           2000      31                                           C        300           2200      11                                           D        350           2000      >100                                         E        380            900      49                                           F        330           2000      26                                           G        340           1800      23                                           H        310            900      17                                           ______________________________________                                    

EXAMPLE 2

A series of preparation were made as in Example 1, except that thedouble amount of glutaraldehyde was used, i.e. 10 ml 50% glutaraldehydebefore addition of polymer (or polyethyleneimine) and 20 ml after. Thetotal amount of glutaraldehyde thus amounts to 36%.

The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Polymer   GI-activity IGIU/g                                                                          Pressure drop, g/cm.sup.2                             ______________________________________                                        Sedipur   220           4                                                     B         200           4                                                     C         290           6                                                     D         260           5                                                     G         240           2                                                     ______________________________________                                    

EXAMPLE 3

A series of preparations were made as in Example 1, except that half ofthe amount of polyethylene imine or the polymer of 1-amino ethylene withor without N-vinyl formamide (2.5% on dry matter) was used. The resultsare shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                 GI-activity Half-life                                                Polymer  IGIU/g      hours     Pressure drop                                  ______________________________________                                        Sedipur  500         1100      148                                            B        480         --        184                                            C        550         1100      356                                            D        540         1200      551                                            G        505         --        127                                            ______________________________________                                    

EXAMPLE 4

1 liter of glucose isomerase containing fermentaiton liquid fromStreptomyces murinus (4% dry substance content) was mixed with 10 g_(MgSO).sbsb.4.7H₂ O and pH adjusted to 7.5. 15 ml 50% glutaraldehydewas added and the cell sludge was stirred for 10 minutes underpH-adjustment to pH 7.5. Then 2.5% (on dry matter) of polymer C and 2.5%(on dry matter) of chitosan (Kayamic 400, a product from Nippon KayakuCo., Ltd.) dissolved in 460 ml 0.5% acetic acid was added and thoroughlymixed. pH was added and thoroughly mixed. pH was constantly adjusted to7.5. After 1 hour the cross-linked mixture was flocculated by additionof a cationic flocculant, Superfloc C521 (Cyanamid Int.). Thecross-linked enzyme was recovered by filtration, formed into particlesby extrusion through a 0.8 mm screen and dried at room temperature.

The results were:

GI-activity IGIU/g: 450

Pressure drop: 89

EXAMPLE 5

A series of preparations were made as in Example 1 except that all theglutaraldehyde was added to the fermentation liquid before the polymerand that the glutaraldehyde concentration was varied.

The results are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                               Glutaraldehyde                                                                             GI-activity                                                                             Half-life                                                                             Pressure drop                           Polymer                                                                              concentration, %                                                                           IGIU/g    hours   g/cm.sup.2                              ______________________________________                                        Sedipur                                                                              18           275       2000     4                                      Sedipur                                                                              27           240       1800     4                                      Sedipur                                                                              36           200       1500     7                                      C      18           300       1700    13                                      C      27           270       1500    20                                      C      36           205       1600    16                                      ______________________________________                                    

The results of the examples show that according to the invention,samples with good activity, stability and physical strength, suitablefor use in fixed-bed column, can be obtained with various compositionsof polymer and with various amounts of glutaraldehyde. Preparations withparticularly good stability and physical strength are obtained withpolymer containing 25-50% 1-amino ethylene. Preparations withparticularly good physical strength are obtained by using 30-40%glutaraldehyde.

We claim:
 1. A particulate immobilized enzyme preparation obtained by aprocess comprising the sequential steps of:a) providing an aqueousmedium comprising an active enzyme, b) adding a polymer selected fromthe group consisting of homopolymers of 1-amino ethylene and copolymersof 1-amino ethylene and N-vinyl formamide, c) adding a cross-linkingagent for amino groups, d) holding the mixture to effect cross-linkingof the enzyme and the polymer with the cross-linking agent, andflocculation to produce a flocculant, e) dewatering the flocculant, f)sub-dividing the dewatered flocculant, and g) drying the sub-dividedflocculant.
 2. The immobilized enzyme preparation according to claim 1,wherein said polymer is added in amount of 2-30% by weight of the drymatter in said aqueous medium.
 3. The immobilized enzyme preparationaccording to claim 1, wherein said copolymer comprises 10 to 100 mole %of --CH₂ --CH(NH₂)-- units and less than 90 mole % of --CH₂--CH(NH--CHO)-- units.
 4. The immobilized enzyme preparation accordingto claim 1, wherein chitosan is added prior to the addition of thecross-linking agent in step c).
 5. The immobilized enzyme preparationaccording to claim 1, wherein the cross-linking agent is glutaraldehyde,polyazetidine or a diisocyanate.
 6. The immobilized enzyme preparationaccording to claim 1, wherein the process comprises the additional stepsof:adding the same cross-linking agent used in step c) to the aqueousmedium of step a) and holding to effect partial cross-linking of theenzyme with the cross-linking agent prior to the addition of the polymerin step b).
 7. The immobilized enzyme preparation according to claim 1,wherein the cross-linking agent is added in an amount of 5-40% by weightof the dry matter in said aqueous medium.
 8. The immobilized enzymepreparation according to claim 1, wherein said aqueous medium is anenzyme solution.
 9. The immobilized enzyme preparation according toclaim 1, wherein said aqueous medium comprises enzymatically activecells or a cell mass made by disruption of said cells.
 10. Theimmobilized enzyme preparation according to claim 1, wherein saidaqueous medium further comprises an enzymatically inactive protein. 11.The immobilized enzyme preparation according to claim 1, wherein theenzyme is a glucose isomerase.
 12. The immobilized enzyme preparationaccording to claim 11, wherein the glucose isomerase is derived from astrain of Streptomyces.
 13. A continuous process for the isomerizationof glucose, comprising passing a glucose solution through a columncontaining a fixed bed of the immobilized glucose isomerase according toclaim
 11. 14. A process for producing an immobilized enzyme preparation,comprising the sequential steps of:a) providing an aqueous mediumcomprising an active enzyme, b) adding a polymer selected from the groupconsisting of homopolymers of 1-amino ethylene and copolymers of 1-aminoethylene and N-vinyl formamide, c) adding a cross-linking agent foramino groups, d) holding the mixture to effect cross-linking of theenzyme and the polymer with the cross-linking agent, and flocculation toproduce a flocculant, e) dewatering the flocculant, f) sub-dividing thedewatered flocculant, and g) drying the sub-divided flocculant.
 15. Theprocess according to claim 14, wherein said polymer is added in anamount of 2-30% by weight of the dry matter in said aqueous medium. 16.The process according to claim 14, wherein said copolymer comprises 10to 100 mole % of --CH₂ --CH(NH₂)-- units and less than 90 mole % of--CH₂ --CH(NH--CHO)-- units.
 17. The process according to claim 14,wherein chitosan is added prior to the addition of the cross-linkingagent in step c).
 18. The process according to claim 14, wherein thecross-linking agent is glutaraldehyde, polyazetidine or a diisocyanate.19. The process according to claim 14, wherein the process comprises theadditional steps of:adding the same cross-linking agent used in step c)to the aqueous medium of step a) and holding to effect partialcross-linking of the enzyme to the cross-linking agent prior to theaddition of the polymer in step b).
 20. The process according to claim14, wherein the cross-linking agent is added in an amount of 5-40% byweight of the dry matter in said aqueous medium.
 21. The processaccording to claim 14, wherein said aqueous medium is an enzymesolution.
 22. The process according to claim 14, wherein said aqueousmedium comprises enzymatically active cells or a cell mass made bydisruption of said cells.
 23. The process according to claim 14, whereinsaid aqueous medium further comprises an enzymatically inactive protein.24. The process according to claim 14, wherein the enzyme is a glucoseisomerase.
 25. The process according to claim 24, wherein the glucoseisomerase is derived from a strain of Streptomyces.